Angiogenesis is one of the primary factors resulting in the growth and metastasis of malignant tumors [1]. The process of angiogenesis is regulated by many factors, among which some factors promote angiogenesis, while some factors inhibit angiogenesis, and as a result, the regulation of angiogenesis is a very complicated dynamic process [2]. Anti-angiogenesis treatment is intended to control the growth of a tumor by blocking angiogenic stimulating factors or preventing angiogenesis in the tumor using angiogenesis inhibitors. At present, a large amount of angiogenic stimulating factors are known, such as, for example, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF) etc. which may stimulate the division and differentiation of vascular endothelial cells and the morphogenesis of blood vessels. Among these factors mentioned above, it is now known that VEGF is the most angiogenesis-specific and the most effective growth factor [3, 4].
In a hypoxic environment inside tumor tissue, VEGFs are secreted by the tumor cells, which induce the division and migration of vascular endotheliocytes, resulting in the establishment of a tumor vascular network. It has been demonstrated that the inhibition of VEGF may prevent angiogenesis, and further inhibit the growth of tumor. For this reason, VEGF and its receptors are important targets for anti-angiogenesis medicaments.
At present, anti-angiogenesis medicaments demonstrated in clinical trials to have efficacy include Bevacizumab (under the trade name of Avastin), which is able to block VEGF directly and inhibit the tumor angiogenesis. Bevacizumab was approved for marketing by the FDA in 2004, and as a first-line drug for rectal cancer, it is the first marketing-approved drug that plays a role in anticarcinogenesis by inhibiting angiogenesis. Avastin is a humanized anti-VEGF monoclonal antibody, which is produced by Genentech. In a large-scale Phase III clinical trial, the combined therapy by Avastin and chemotherapy may significantly extend the survival time of the patients suffered from many kinds of cancers, including rectal cancer, lung cancer, breast cancer and renal cancer. [5, 6] The clinical success of Avastin is a landmark, demonstrating that the anti-angiogenesis treatment using tumor vascular system as the target is a clinically effective measure and provide a new path for the tumor treatment.
Besides Avastin, several drugs for anti-VEGF signaling are also in the late phase of human clinical trial and are expected for clinical application in the next several years. Among others, Aflibercept (also called as VEGF-Trap), developed by the Regeneron and Sanofi-Aventis, is now in Phase III clinical trial[7]. An anti-VEGF receptor II (VEGFR2) monoclonal antibody drug IMC-1121B (Imclone) is also in Phase III clinical trial[8]. The amino acid sequence of VEGFR2 is provided in SEQ ID NO: 3 and the nucleotide sequence of VEGFR2 mRNA is provided in SEQ ID NO: 6.
Great progress has been achieved in the clinical treatment of tumor using anti-VEGF medicament, however, it has also been shown by the clinical trial that the anti-VEGF treatment are also considerably limited. From the point of the effect of tumor treatment, Avastin may extend the half survival time of the colon cancer patient for about 3-4 months [9, 10], and extend the half survival time of the breast cancer patient for about 7-8 months [11], and thus, Avastin cannot effectively inhibit the growth of tumor blood vessel over the long term.
The primary causes resulting in the failure of anti-VEGF treatment or the appearance of resistance may depend on the regulation of tumor angiogenesis by a plurality of factors. Although VEGF plays an important role in angiogenesis, it is not the only angiogenesis stimulating factor. Meanwhile, owing to the heterogeneity of tumor cells, the complexity of tumor microenvironment and the compensatory response mechanism of body, when the activity of VEGF is inhibited for a long period of time, other angiogenesis stimulating factors would be expressed [12], and thus the growth of tumor blood vessel is no longer dependent on VEGF signaling path.
The variation of angiogenesis factors expressed by the tumor was studied during anti-VEGFR2 treatment for pancreatic tumor by Prof Hanahan's group (University of California, San Francisco, US), indicating that the expression of several genes changed during anti-VEGF treatment, in which the expression of FGF-2 significantly increased. It has been shown that the expression of FGF, especially FGF-2, increased significantly in the tumor resistant to anti-VEGF treatment so that angiogenesis was activated again and the tumor repopulation was inhibited after blocking FGF signal pathway [13]. It may be seen that the over-expression of FGF-2 is closely related to the ability of tumor to escape from anti-VEGF treatment.
Fibroblast growth factor (FGF) is a growth factor family for heparin-binding, and there are 22 family members (FGF 1-14, 16-23) in mammals. FGF plays an important role in many biological functions, for example, cell proliferation, differentiation, migration, angiogenesis and tumorigenesis. Fibroblast growth factor receptor (FGFR) is the receptor that binds the family members of fibroblast growth factor. FGF may bind FGFR and activate the downstream signal pathway, which plays an important role in a physiological and pathological process, such as embryogenesis, development, vasculogenesis, vasodilatation, neuroregulation, ischemia protection, wound healing and tumorigenesis. [14, 15] It has been demonstrated that overexpression of FGF/FGFR in vivo is closely related to many diseases including tumors (such as fibroma, neuroglioma, melanoma, prostate carcinoma, lymphomata, leukaemia, urinary, and system cancer), skeletal system diseases (dwarfism, craniosynostosis, achondroplasia, and acanthosis nigricans) and renal failure. It has been reported that increased expression level of FGF and its receptor may directly promote the survival and proliferation of tumor cells, and the survival of hepatic carcinoma cells is significantly reduced by down-regulation of FGF by siRNA [22].
At present, few researches focus on the development of new anti-angiogenesis medicament using FGF and its receptor as the target in clinical trials. For example, FP-1039, a fusion protein composed of whole extracellular domain of human FGFR1 and human IgG1 Fc fragment, is developed by a US company Five Prime and now in volunteer recruitment stage of Phase I clinical trail. However, it has been suggested by researches of Wang and Olsen that the first Ig-like domain of the extracellular domain of human FGFR1 and the linking fragment between the first and the second Ig-like domain of the extracellular domain of human FGFR1 may inhibit binding of FGFR1 and FGF [20, 21].
The tertiary structure of a protein is closely related to its biological function. The FGF binding capacity is directly influenced differences among the conformations of each Ig-like domain of the extracellular domain of FGFR and the linking fragment. Different fusion proteins, composed of the FGFR extracellular domain fragments of various lengths and IgG Fc, are constructed by means of genetic engineering to obtain fusion proteins with different conformations, so that the fusion protein with high efficiency of FGF binding and biological activity can be screened.
There are four FGFR genes in mammals: fgfR1-fgfR4. Fibroblast growth factor receptor is composed of the extracellular domain, transmembrane domain and intracellular domain. There are many members in FGFR family, which have similar extracellular domain but vary in the ligand binding property and kinase domain. Their extracellular domains include three immunoglobulin-like (Ig-like) domains: the first Ig-like domain, the second Ig-like domain and the third Ig-like domain, and there is a sequence between the first and the second Ig-like domain, which is referred as the intermediate functional sequence of the Ig-like domain of FGFR (IFS for short herein) in this specification. The intermediate functional sequence may comprise one acidic amino acid segment, which is referred as acidic box (AB).